Lipoic acid is an essential sulfur-containing cofacor that is found in several multienzyme complexes that are involved in energy metabolism. In its functional form it is covaleritly attached to the epsilon amino group of a specific lysine residue on a designated lipoyl carrying protein. The detailed manner in which it functions in these complexes has been known for quite some time, and is found in almost every beginning biochemistry textbook. By contrast, only within the last few years has a clear understanding of its biosynthesis begun to emerge. Lipoic acid is biosynthesized in its cofactor form rather than its free acid form, requiring two proteins that are dedicated to the transformation. The first, octanoyl transferase (LipB) catalyzes the transfer of the eight-carbon fatty acyl chain from octanoyl-ACP to one of the several lipoyl carrying proteins in the cell. The second protein, lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at positions 6 and 8 of the fatty acyl group, affording the lipoyl cofactor. LipA is a member of radical SAM superfamily of enzymes, which use a [4Fe-4S] and S-adenosylmethionine to generate high-energy radicals that are intermediates in each reaction. The long-term objective of this proposal is to understand at the detailed molecular level how each of these enzymes work. Particular focus will be directed at LipA, to understand exactly from where the sulfur atom that is inserted into the substrate is derived. Efforts will involve bacterial genetics, transient state kinetics, and spectroscopy (UV-vis, EPR, M"ssbauer, ENDOR, and FT-ICR mass spectrometry). Aside from the involvement of lipoic acid as a cofactor in enzyme complexes of energy metabolism it is known to modulate glucose metabolism in patients with type II diabetes and to serve as a general cellular antioxidant, among many other things. There is significant evidence that lipoic acid can be endogenously synthesized in mammalian cells, and that the inhibition of this pathway compromises cellular function and leads to death.